Fig. 3
Characterization of a 3D B cell follicular organoid to model the GC reaction. a Scheme of 3D B cell follicular organoid fabrication. Splenic B cells are co-encapsulated with 40LB stromal cells and IL4 into an RGD-presenting nanocomposite hydrogel that contain gelatin ionically cross-linked with synthetic silicate nanoparticles. b Representative fluorescence pictures of splenic GFP B cells in 3D organoid culture. c Flow cytometry plot of a 3D B cell follicular organoid. The gated area on the top shows the live B cells (B220 + DAPI−) and on the bottom plot, the organoid GC B cells (GL7 + FAS + ) within live B cells. d Average of GC B populations of organoids quantified as in c. e Average of percentage of proliferating organoid GC B populations, quantified as indicated in Supplementary Fig. 4B. f MFI of proliferation dye from e. g Flow cytometry plot of GC B cells stained with annexinV. h Average of percentage of apoptotic GC B cells quantified as in g. i RNA-seq profiles of organoid GC B cells after 4 and 6 days in culture (organoid GCB d4, n = 4 spleens, and d6, n = 3 spleens) were projected into the principal component space defined by GC B cells sorted from immunized mice (in vivo GCB, n = 6 mice), CD138 + plasma cells (in vivo PC, n = 6 mice) and FAS-GL7-IgD + B220 + naive B cells (NB, n = 3 mice). j, k Heat maps of gene expression level of GC B cells showed in i, represented as log2 ratio relative to mean naive B cells j and to mean plasma cells k. PC1/2 = principal component 1/2. l Splenocytes were permeabilized and stained for EZH2, and co-stained for GL7, FAS, IgD and B220 to identify GC B cells (FAS + GL7 + B220 + ) and naive B cells (FAS-GL7-IgD + B220 + ). m MFI of EZH2 (n = 3 per group) from l. n, o The indicated immunoglobulin variable loci were sequenced from organoid or in vivo GC B and naive B cells (n = 3 biological replicates per group). t test vs. naive B cells, *P < 0.05, **P < 0.01. Values in d–f, h, m–o are mean ± SEM. d–h, n = 3 organoids per time-point. Results shown in b–h are representative of 4 independent experiments. See also Supplementary Fig. 4
